Hitherto, determination of DNA has placed great importance on detection of a DNA that has a specific nucleotide sequence, and little effort has actually been put into developments of techniques for determining the amount of double-stranded DNA in a sample in a simple and easy way with precision.
Although an absorbance ratio (260/280 nm) in the ultraviolet region is often used for simple determination of DNA, it is greatly influenced by impurities and nucleic acid components other than double-stranded DNA, and thus is low in accuracy for determining double-stranded DNA.
Meanwhile, methods for determining double-stranded DNA only are usually those that measure changes in fluorescence intensity of a substance capable of specifically binding to double-stranded DNA. However, they are problematic in that their background is high and measurable concentration range is narrow, thereby rendering processing of measurement data to be complicated.
In addition to such conventional simple determination methods, methods for determining double-stranded DNA utilizing an electrochemical technique have been developed. For example, there are a method in which a double-stranded DNA is supplemented with a substance capable of specifically binding to the double-stranded DNA so as to detect electrochemical behavior of a complex of the double-stranded DNA and the double-stranded DNA binding substance, and a method in which the amount of the residual double-stranded DNA binding substance which fails to bind to the double-stranded DNA is electrochemically determined (see, for example, Patent Documents 1 and 2).
According to the method disclosed in Patent Document 1, an excess amount of the double-stranded DNA binding substance is added to a sample containing a double-stranded DNA, and then an amount of electrode oxidation current for the free DNA binding substance which fails to bind to the double-stranded DNA is determined so as to obtain the amount of double-stranded DNA. This method requires a high-level technique for manufacturing an electrode since the electrode must always keep the surface area thereof constant with precision so as to detect a weak oxidation current.
According to the method disclosed in Patent Document 2, an immobilized enzyme electrode is used in order to amplify a weak oxidation current on the electrode. However, this method still requires a high-level technique for manufacturing an electrode, and is still problematic in that it requires a sample solution to have a complicated composition since the oxidation-reduction reaction catalyzed by the immobilized enzyme must be conjugated with another reaction so that the reactions proceed reversibly.
Patent Document 1: Japanese Patent Laid-Open No. 2002-218998
Patent Document 2: Japanese Patent Laid-Open No. 2004-257993